Isolated Codon Sequence

ABSTRACT

The present invention is a method for the biosynthesis of hundreds of compounds, mainly found in the Cannabis plant. The starting material for these compounds can be any biological compound that is used/produced in a biological organism from the sugar family starting materials or other low cost raw materials processed via enzymes or within organisms to give final products. These final products include, but are not limited to: cannabinoids, terpenoids, stilbenoids, flavonoids, phenolic amides, lignanamides, spermidine alkaloids, and phenylpropanoids. Specifically, the present invention relates to the regular/modified/synthetic gene(s) of select enzymes are processed and inserted into an expression system (vector, cosmid, BAC, YAC, phage, etc.) to produce modified hosts. The modified host is then optimized for efficient production and yield via manipulation, silencing, and amplifying inserted or other genes in the host, leading to an efficient system for product.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 15/719,430,filed on Sep. 28, 2017 entitled “An Isolated Codon Optimized NucleicAcid”, which is continuation of U.S. Ser. No. 15/096,164, filed Apr. 11,2016, entitled “A Novel Method for the Cheap, Efficient, and EffectiveProduction of Pharmaceutical and Therapeutic API's, Intermediate, andFinal Products”, that claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/145,430, entitled “A Novel Method for the Cheap,Efficient, and Effective Production of Pharmaceutical and TherapeuticAPI's, Intermediate, and Final Products”, filed Apr. 9, 2015, both ofwhich are herein incorporated by reference in their entirety for allpurposes.

The Sequence Listing, which is a part of U.S. Ser. No. 15/719,430, filedon Sep. 28, 2017 entitled “An Isolated Codon Optimized Nucleic Acid”,includes a computer readable form and a written sequence listingcomprising nucleotide and/or amino acid sequences of the presentinvention. The sequence listing information recorded in computerreadable form is identical to the written sequence listing. The subjectmatter of the Sequence Listing is incorporated herein by reference inits entirety.

FIELD OF INVENTION

The present invention is in the technical field of large scaleproduction of pharmaceutical and supplemental products for variouscommon illnesses, medical conditions, and general industrial use. Moreparticularly, the present invention is in the technical field ofbio-synthesis of cannabinoids, terpenoids, stilbenoids, flavonoids,phenolic amides, lignanamides, spermidine alkaloids, andphenylpropanoids; compounds found in Cannabis sativa, along with variouscombinations and specialized formulations which are beneficial inailments ranging from cancer to glaucoma. The final product(s) can be anintermediate or a compound of interest. The core concept of theinvention is based on the idea of cheaper and more efficient production,along with novel products and applications.

INTRODUCTION

Cannabinoids from Cannabis have been used for thousands of years fortreatment of various ailments and conditions in many different culturesaround the world. However, most of various types of cannabinoids inCannabis are at a very low concentration in the plant. Therefore, mostpatients/users never get a threshold dosage for any kind of relief fromanything other than tetrahydrocannabinolic acid (THC/A),cannabinolic-acid (CBD/A), and cannabinol (CBN). There are a few strainsor concentrates available that have a rare cannabinoid, but are usuallyvery highly concentrated in tetrahydrocannabinol (THC) or cannabidiol(CBD) to have any pronounced effect by the rare cannabinoid.

In other words, the pharmaceutical industry has not tapped into the realpotential of the Cannabis plant. With time, more research is beingconducted into the different kinds of cannabinoids and their medicinalapplications. Researchers are finding that many of the othercannabinoids also have unique medicinal properties.

SUMMARY

Biosynthesis of important molecules can be used for therapeuticapplications, bulk substance production, intermediate API biosynthesis,and various other novel formulations and applications for suchsubstances, as known to those skilled in the art. Many biologicalmolecules can be changed/converted into molecules of importance by usingenzymes and other processes. This process can be utilized by employingmethods for transforming a range of starting materials into finalproducts to be used in pharmaceuticals and supplements as activeingredients, or donating a significant portion of their structure to thefinal active ingredients. The final products can also be used in otherindustries and applications, such as food, beverage, and other goodsproduction. For example, table sugar, starch, and cellulose can beconverted to glucose, creating a molecule that can readily be utilizedby any organism as an energy source. Therefore, depending on thespecific compound(s) being manufactured, and the kind(s) of startingmaterials available, along with the host and production technique(s) anykind of host engineering, various expression systems and methods, andvarying materials, a spectrum of different methods and products ispossible.

The advantages of the present invention include, without limitation,creation of hundreds of compounds from readily available biologicalmolecules that can be produced and harvested from virtually all knownsources of plants and other energy producing organisms. Since sugarproducing plants and organisms, biomass, and carbon based industrialwaste products are very abundant, our “raw material” will be very cheapand easy to obtain anywhere in the world. After scaling up the givenmethods, hundreds of compounds with medicinal properties can be producedat a very low cost, allowing the widespread distribution and aiding ofmillions of people.

Another advantage is that there is no need or use of growing any illegalplants. For example, no marijuana, poppy, or other plant production isnecessary. This is advantageous as it will lead to drastically cuttingdown the production, consumption, and trafficking of many unregulatedsubstances.

The most important advantage of the present invention is that we canmake and use many compounds that are virtually so low in concentrationin the Cannabis plant, that there is no effect in using Cannabis if weare only after the therapeutic effects of these compounds. For example,patients using marijuana can only benefit from tetrahydrocannabinolicacic (THCA), THC, cannabidiolic acid (CBDA), CBD, CBN, and a few othercompound class families, as the concentrations of the other compounds isso low that it has no effect. This invention will allow the productionof hundreds of compounds in pure form, leading to many new medicaldiscoveries and applications.

BRIEF DESCRIPTION OF THE FIGURES

The nature, objects, and advantages of the present invention will becomemore apparent to those skilled in the art after considering thefollowing detailed description in connection with the accompanyingfigures, in which like reference numerals designate like partsthroughout, and wherein:

FIG. 1 is a diagram of the pathway for the biosynthesis of all moleculesof interest via the conversion of starting materials to glucose and thento final products;

FIG. 2 is a diagram of the pathway for the biosynthesis of cannabinoids;

FIG. 3 is a diagram of the pathway for the biosynthesis of stilbenoids;

FIG. 4 is a diagram of the pathway for the biosynthesis ofphenylpropanoids and flavonoids;

FIG. 5 is a diagram of the pathway for the biosynthesis of phenolicamides and ligananamides;

FIG. 6 is a diagram of the pathway for the biosynthesis of spermidinealkaloids;

FIG. 7 is a diagram of the combined biosynthetic pathways of FIGS. 1-6;and

FIG. 8 is diagram of the genetic modification of certain genes forhigher product yield in Saccharomyces cerevisiae yeast.

DETAILED DESCRIPTION

The present invention is a method for the biosynthesis of hundreds ofcompounds, mainly found in the Cannabis plant. The starting material forthese compounds can be any biological compound that is used/produced ina biological organism from the sugar family starting materials or otherlow cost raw materials processed via enzymes or within organisms to givefinal products. These final products include, but are not limited to:cannabinoids, terpenoids, stilbenoids, flavonoids, phenolic amides,lignanamides, spermidine alkaloids, and phenylpropanoids (collectively,“final products”).

Definitions, Terms, Elements

The Following are a List and their Definitions:

Genetic engineering: targeted manipulation of a cell's geneticinformation;

Rational Metabolic Engineering: engineering of enzymes, transporters, orregulatory proteins based on available information about enzymes,pathways, and their regulation.

Evolutionary engineering: encompasses all methods for empirical strainimprovement (mutagenesis [natural or induced] and recombination and/orshuffling of genes, pathways, and even whole cells; usually performed incycles or sequentially

Cannabinoids: compounds that are terpenophenolic with 22 carbons (21carbons for neutral forms), found in Cannabis

Terpenoids: also known as isoprenoids, class of organic compounds

Stilbenoids: hydroxylated derivatives of stilbene

Flavonoids/phenylpropanoids: compounds derived from or usingphenylalanine as a precursor

Lignanamides/phenolic amides: compounds produced through tyraminepathways

Spermidine alkaloids: compounds produced through glutamic acid pathways

Starting material/reactant/excipient: compounds used for the initialstep of biosynthesis, which are cheap and readily available

Intermediate: products that are formed within the biosynthesis pathways,which can further be processed to make final products, or can,themselves, be utilized as a final product

Final product/product/end product/compounds of interest: cannabinoids,terpenoids, stilbenoids, flavonoids, phenolic amides, lignanamides,spermidine alkaloids, and phenylpropanoids

In-vivo: inside the cell

In-vitro: outside the cell

BAC: bacterial artificial chromosome, carrier of DNA of interest intohost

YAC: yeast artificial chromosome, carrier of DNA of interest into host

Vector/cosmid/phage: carrier of DNA of interest into host

Starting Materials

All biological organisms produce organic molecules that are processed inmany different processes in the organism. The present invention utilizesstarting materials that are either:

1) Readily available and relatively pure

2) Cheap to produce or buy

3) Easily modified (via enzymes, catalysts, or other methods)

Based on the above criteria, there are multiple groups and families ofcompounds that would fit one or all three of the above criteria. Thesegroups and families of compounds include, but are not limited to:ligno-cellulosic biomass, forest biomass, energy/food production waste,but are not limited to: ligno-cellulosic biomass, forest biomass,energy/food production waste, commonly available sugar-based substrates,food and feed grains.

Sugars and metabolic intermediates from cellular processes can be usedas starting materials. Sugars can be found in abundance in manysubstances, including, but not limited to the following: rice,soya/rape, cereals (maize), wheat, beans, sugar beet (sugar cane), plantbiomass (wood), grasses, and various other sources. Starch, cellulose,fructose, ethanol, and saccharose in the aforementioned substances canbe enzymatically converted to glucose, which, after filtration andpurification steps, can be used as a raw material for the finalproducts.

Subsequent steps can also be performed on the lignocellulose, whichfurther makes hemicellulose and cellulose, both which make glucose. Anadvantage of this method is that there are by-products generated whichcan be sold as raw material to make hydrocarbons, biogas, and other fuelsources. Whole crops or parts of crops, or waste matter from cropproducts can be used and incorporated into this system, yielding an“eco-friendly” facility. Products made from these raw materials can useany of the starting materials listed in Table 2.

Within the realm of readily available non-biomass/crop bulk material,HFCS (high fructose corn syrup) is a cost effective syrup made withfruit sources that contains anywhere from 30-90% fructose, along withsome other sugars. Plants that make molasses, HFCS, and other sugars canbe genetically modified to enhance the production of sugar, leading tobetter yields of starting material from the crop. Other products fromthese plants can also be incorporated into compounds of interestproduction via slight system modification. Biodiesel, ethanol, glycerol,lactic acid, whey and glucose are a few others. These work due to thefact that any of these products can be converted into starting materialfor our own purposes using enzymatic or physiochemical tools.

Plants also have their own innate levels of metabolites that can beharvested into the process from a plant biomass source. Processes can becrafted that utilize most of the metabolites and biomass for APIproduction giving the maximum efficiency and usability per amount ofstarting material used. (Enzyme combinations or chambers that utilizemost intermediates, sugars, oils, etc. in each biomass load).

Biorefineries can be custom designed that cater to specific raw material(plant biomass for harvesting lignocellulose which is further processedand refined into a simple carbohydrate used in the API manufacturingprocesses). During certain steps throughout the process, thermochemicaland other processing can be used for higher efficiencies which are notpossible with biochemical processing alone.

Another group of cheap starting materials is agricultural residue,grass, aquatic biomass, and water hyacinth. Products such as oils andalcohols can be made with these bulk materials. These materials can beconverted enzymatically and chemically into starting materials that canreadily by injected into our API production system.

Specifically, biorefineries can be designed to be extremely efficient,using all parts of the raw material. For example, concerning plantbiomass, the biomass can be step-wise processed so we are able toharvest all individual components. The first step can be using solventto extract terpenes, alkaloids, etc. Other methods can be used toextract steroids, triglycerides, and other valuable metabolites. Finallythe biomass can be treated with cellulases to give glucose, which is oneof the primary raw materials of choice.

Production Roadmap Summary

The present invention is a method that covers the bio-synthesis ofhundreds of compounds, mainly found in the Cannabis plant. The startingmaterial for these compounds can be any biological compound that isused/produced in a biological organism from the sugar family startingmaterials or other low cost raw materials processed via enzymes orwithin organisms to give final products. Information related to thestarting materials were detailed in the previous section.

Most sugars and related compounds can be inter-changed using variousenzyme systems. For example, we can convert glucose to fructose usingFructose 6-Phosphate (F-6-P) as an intermediate.

Apart from starting materials, we can either:

1) Make enzymes via vectors in bacteria (e.g. E. coli) or yeast (e.g. S.cerevisiae), extract enzymes, and create in vitro models for makingcannabinoids.

2) Make enzymes via protein synthesizing systems (Protein Synth. Robot,Cell Free Expression Systems, etc.)

3) Make final products (compounds of interest) in bacteria or yeast viavectors, plasmids, cosmids, mRNA, various RNA, etc; feed them substrateand purify product.

4) Genetically engineer strains of bacteria and yeast that specialize incannabinoid production, or intermediate production, or substrateproduction, etc.

5) Use organic chemistry for certain parts of the above processes.

6) Use various plant starting material for large quantities ofsubstrates or intermediates.

7) Genetically engineer various plants to produce cannabinoids. (e.g.Tomatoes or celery that naturally produce cannabinoids, or algae thatproduces cannabinoids)

8) Using bioengineered or unengineered C. sativa or any otherplant/algae cell lines for enzyme/substrate/intermediates/product(s)production.

9) Protein engineering on the various proteins involved in theprocesses; engineering will enhance the functionality, ruggedness, andefficiency of the enzymes, and altering them into a novel protein, onenot found to be covered in any of the above prior art patents.

10) Genetically engineer various plant species to produce higheryielding raw material (sugars) to be used in production of the products.A possibility is to have an indoor/grow for different plants to be usedas raw material producers.

After the final product is made, a purification system will filter andconcentrate the target molecules. Examples include large scalefiltration systems such as chromatography. Once a pure product, we canutilize liquid solutions, caps, sprays, and other delivery systems.

As many of these final products are made, their applications can be seenfrom glaucoma to cancer, or general well-being. Certain cofactors can becombined with certain final products for more efficacy against specificmedical conditions (e.g. combine certain vitamins or other therapeuticcompounds with certain compounds of interest). We can also make finalproducts that have certain combinations of compounds of interest withother cofactors as well (e.g. combine THCA/CBDA/Vitamin C, orCBDVA/CBD). This patent covers all the products above and also onesdiscovered in the future based on the same principles and methods.

DETAILED DESCRIPTION OF THE FIGURES

Referring now to the invention in more detail, in FIG. 1 there is showna family of sugars and other common derivatives. Along each arrow foreach reaction, the number denotes a specific enzyme that catalyzes thereaction. Starting with any sugar in FIG. 1 (list of starting materialsin Table 1), we can convert it to glucose to incorporate it into thereaction using the appropriate enzyme, as known to those skilled in theart. An unlimited number of ways are possible when dealing with anystarting material, as described above. Enzymes needed for each kind ofsubstrate can be made in vivo or in vitro just as we will be doing forthe enzymes in the final product or intermediate production. The finalsugar that enters our mechanism will be either glucose or fructose.Through the glycolysis pathway, the sugar will be converted intoAcetyl-CoA with the addition of ATP and CoA (shown in FIG. 1). From thispoint on, the intermediate can follow a variety of paths that can leadto hundreds of products. There are many alternative ways of doing this.We can use the DOX, MEP or MVA pathways to get IPP and DMAPP, which giveus GPP and NPP. For a reaction with Olivetolic Acid or Divarinolic Acid,we get many cannabinoids as final products.

The generalized pathway for the production of cannabinoids once thestarting material is converted to glucose is the following, usingappropriate enzymes as known by those skilled in the art:

Glucose→Fructose→F-6-P→F1:6BP→3-P-Glyceraldehyde→1,3-BPG→3PGA→2-PGA→PEP→Pyruvate→Acetyl-CoA→AcetoacetylCoA→HMG-CoA→MVA→MevalonicAcid→Mevalonate-5-P→Mevalonate-5-PP→Isopentyl-5-PP→Dimethylallyl-PP→NPP/GPP→GPP

This general pathway is outlined in FIG. 1. From this point on, thepathway can utilize Olivetolic Acid or Divarinolic Acid with GPP,yielding CBGA or CBGVA, which can further yield other cannabinoids, asshown in FIG. 2.

The pathways for stilbenoids, phenylpropanoids, and flavonoids work in asimilar fashion. Phenylalanine is generated from sugars, which is thenfurther processed into other compounds using enzymes to final compounds,as shown in FIG. 3 and FIG. 4.

Phenolic amides and lignanamide pathways are derived from tyraminemolecules reacting with other compounds, as shown in FIG. 5. Tyraminecan also be synthesized in our cells of interest as most livingorganisms contain the pathway to synthesize tyramine on their own. Sameis the case for spermidine alkaloid production, as most cells alreadyproduce glutamic acid, which can be further processed by enzymes intothe final components, as shown in FIG. 6.

FIG. 7 is the total pathway overview, showing how all the differentclasses of compounds can be made, and the general paths they take forbeing biosynthesized in the cell.

Overview of Procedure

A general scheme of the work flow is as follows:

1) Regular/modified/synthetic gene(s) of select enzymes are processedand inserted into an expression system (vector, cosmid, BAC, YAC, phage,etc.) to produce modified hosts.

2) Mod host is then optimized for efficient production and yield viamanipulation, silencing, and amplifying inserted or other genes in thehost, leading to an efficient system for product. It is important toremember that every organism is different, and to get a specificcompound each optimization will also be different.

3) Mod host can produce enzymes and final products/intermediates, or befurther modified using host engineering techniques. (Host engineeringCan also be performed before insertion of exp. System)

4) Mod and engineering hosts produce products and intermediates.

5) Product is purified and can be further modified/processed.

In Table 1, different final products are listed along with possibleuses. This list is by no means exhaustive, and as such this patentcovers any molecules that are made this way. Table 2 lists all possiblestarting materials that can be utilized for a cheap and efficientbiosynthesis.

In more detail, referring to the inter-conversion of sugars, we employenzymes readily available in the market. Pure enzyme stock can bediluted and added to a solution with the substrates. Once the reactionis complete, we can filter out the enzyme via dialysis tubing, byprecipitation out of the solution, chromatography, or other industrialmethods for filtration and purification. Each step in FIGS. 1 to 7 willgive work with this strategy, leading us up to the final products or keyintermediate molecules. Certain steps in the process can be worked on byusing chemical and physical methods as well. For example, prenylation ofcertain compounds can be done outside the cell, as it may beadvantageous to do so since unprenylated compounds are also high valuecompounds. Small batches can be prenylated accordingly to demand via achemical process.

There are also commercially available cell free expression systems,which are able to produce proteins without the need of any host. Withappropriate optimization steps, it is possible to get a cheap andefficient process for production of these compounds using identifiedstarting molecules.

Application Techniques

Referring to bacterial, yeast, plant, and algae incorporation of genes,there are a number of strategies that can be applied to achieve this. Wecan:

1) Add genes for 1-10 enzymes in various commercially available vectors,cosmids, plasmids, etc. Only need 1-10 enzymes added, as others arealready built in most living organisms. For example, glycolysis pathwayand related enzymes are already present in most hosts.

2) Bioengineer genes for better yield and suitability in the host.

3) Bioengineer strains of bacteria and yeast that are specialized inproducing important molecules. Many metabolic strategies exist, withidentification by appropriate screening methods:

1) Rational metabolic engineering: engineering pathways using availableinformation

2) Evolutionary engineering: using random genetic perturbations and/ormutations (via random mutagenesis in whole genome and/or parts)

3) Transposon mutagenesis & gene overexpression libraries:overexpression and/or deletion of single or multiple genes;

4) Global transcription machinery engineering: basal transcriptionfactors mutagenesis causing a global reprogramming of gene transcriptionand/or translation One strategy is to suppress any pathway that is notessential to our goals or the survival of the host. Another is toenhance our key pathways, or mixing and matching the two methods. Thesecond strategy is through rapid directed evolution, possible byproducing many generations so eventually we get a generation of hostthat has evolved with our genes/functions of interest.

4) Bioengineer custom basic life forms that are specifically making ourproducts, using another organism or using synthetic/modifications.Components from other hosts and system to make a custom organism.

5) Bioengineer bacteria and yeast to have enzyme genes in theirchromosomes, and make intermediates or final products inside the host.The product of this process can further be modified.

6) Propagate various colonies of organisms which co-exist symbiotically,with the first making our starting material after utilizing a precursor,and the other colonies making our final product. This process can alsobe incorporated into an ecosystem type setup of different chambers, eachholding different organisms that use specific parts of the raw materialto produce intermediates or final products that can be modifiedpost-manufacturing.

Referring to the extraction of enzymes once they have been produced inthe host, there are many ways to isolate and purify our enzymes. Manyorganisms have the ability to excrete proteins, which can be collectedmuch easier than cell lysis, as known by those skilled in the art. Thistechnique is the preferred method.

Another method is to lyse the host culture and purify with traditionalbiochemistry methods (gels, centrifugation, ammonium sulfateprecipitation, etc.), use a specialized nickel column with a prep HPLC(need to add a HIS tag to our proteins; remove HIS tag afterpurification), etc.

Example 1 (Bacterial)

Bacteria (E. Coli, etc.) are inserted with exp. system giving us amodified host. The mod host can either be further processed or it cangenerate products. Products/intermediates are made in the host, and maybe either enzymes that are further extracted and used in vitro, or weadd substrates into the bacterial culture so they use the enzymesproduced in them to make the substrate. Either way (protein or prodproduction), purification is carried out to get final products, orintermediates that can be further processed in vitro to give finalproducts. Throughout this procedure, host engineering can be carried outat any step of any process to get better yields.

Example 2 (Plants)

Plant tissue can be used as a starting material to get a tissue culturegoing. Appropriate expression vectors/systems carry our interest genesinto the cells. Alternatively, cell engineering can lead to manycombinations that may have similar or different outcomes. The culturecan be grown into full plants, and products are ingested by consumingthe plants (e.g. tomatoes with certain cannabinoids produced within,etc.). The second way uses the cell culture in a synthetic environmentto produce final products/intermediates. Finally, product is purifiedand used.

Example 3 (Algae)

Algae are modified with the usual techniques used for host engineering.Once completed, the mod host can be embedded into a system similar tobiofuel production from algae. Using sunlight and some nutrients, thealgae produces final products/intermediates, which is appropriatelyfiltered from the bulk. Other products generated can be furtherprocessed to get biofuels or other important compounds that can readilybe sold in the market.

Example 4 (Fungi)

Fungi modified with the techniques can:

1) Use plastic to produce final products/intermediates. Plastic needs tobe processed and broken down into components before being used in thisprocess via chemical and biological processes, known by those skilled inthe art.

2) Clean up waste, whilst producing final products/intermediates at thesame time.

3) Produce beer and wine with fungi that also makes finalprod/intermediates. Beer and wine will contain our compounds ofinterest.

4) Use fungi cultures to produce compounds of interest.

5) Genes for S. cerevisiae strains to be modified for better yields offinal products:

tHMGR

upc2-1 (allows higher uptake of exogenous sterol five-fold from medium)

ERG genes (ERG6, ERG2, ERG3, ERG1, ERG11, ERG24, ERG25, ERG9, ERG10,ERG13, ERG12, ERGS, ERG19, ERG20)

HMGR1 and HMGR2

IDI genes

Ga180p

DPP1, ADH2, and ALD6 genes

FPP/GPP synthase (chose avian FPP synthase as it exhibits highercatalytic turnover rates and lower Kms for substrates than otherprenyltransferases)

Manipulation, deletion, overexpression, and other modifications to thegenes listed above will produce strains that are highly efficient forthe production of our compounds of interest. These strains have anexogenous sterol uptake, as the internal sterol pathway has beendisabled by manipulations so that all the carbon flux can be directedtoward the production of our compounds of interest. Example of geneticpathway regulation in yeast is shown in FIG. 8.

Our initial strategy in S. cerevisiae was to increase the carbon flux ofour pathways of interest, while decreasing or eliminating pathways thatled carbon flux away from our pathways as well. We also focused onexogenous sterol uptake for higher production and secretion levels, cellpermeability for more efficient and cheaper production, along withfocusing the pathways on utilizing the cheapest sugars. Dynamic controlover ergosterol regulation can increase yields as well. Overall resultis a strain that is has increased yield many fold, while making theoverall production more stable and cheaper.

1) Perform EMS mutagenesis on yeast strains (BY4741, BY4742, CEN.PK,CEN.PK2, EPY300) to get colonies with a SUE (sterol uptake exogenous)mutation. This enables us to provide exogenous sterol to the yeast whilecancelling out the gene that diverts carbon flux towards ergosterol,thereby increasing total carbon flux. Without the SUE mutation, the celldiverts lots of carbon flux toward manufacturing sterols, therebydiverting the pools of intermediates away from our compounds andinterest leading to very low yields.

2) Perform ERG1 and ERG9 gene knockouts. ERG1 knockout stops theactivity of conversion of squalene to squalene epoxide, therebycomplementing the SUE mutation and allowing higher uptake of exogenousergosterol, while ERGS knockout takes out the cells ability to divertcarbon flux towards other metabolites.

3) On some lines, we can perform a DPP1 knockout. DPP1 knockout ensuresthat FPP/GPP are not converted to FOH, thereby blocking the pathwaytowards FOH products in the cell.

4) Perform ERG2, ERG3, or ERG6 mutations in different cell lines, whileperforming upregulation mutation on upc2-1 gene (general transcriptionfactor) on all three lines. This helps increase cell membranepermeability for better excretion of our compounds without the need forcell lysis and having the ability to use two-phase or continuousfermentation. This also allows the cells to uptake more fatty acids,thereby increasing the yield many fold.

5) Overexpression of ERG10, ERG13, HMGR1/2 or tHMGR, ERG12, ERGS, IDILHFA1 genes in yeast inserted via vectors. By overexpression of thesegenes, we are amplifying the enzymes of the MVA pathway from the sugarsto our compounds, thereby amplifying the intermediates and finalproducts.

6) Modification of avian and/or salmonella ERG20 gene encoded FPPsynthase (ERG20p). Some cells lines can also be modified using theErg20p(F96C) mutations. This allows for higher Kms and increasedcatalytic turnover compared to endogenous GPP synthase, while theengineering itself allows for production of GPP.

7) Ga180p gene deletion so we do not need to use galactose sugar wheninducing promoter expression. This is important since others have usedgalactose promoters, which need expensive galactose sugars forproduction. By deleting this gene, the cells bypass the need forgalactose to express enzymes, leading to cheaper and more efficientbiosynthesis.

8) Adding ADH2p promoter to induce strong transcription under conditionswith low glucose. This promoter is more efficient than the GAL promoter,and has best results while using non-glucose sugars (ethanol, fructose,etc.) which are cheaper.

9) On some lines, we also overexpress ADH2 and ALD6 genes, along withoverexpression of an acetyl-CoA C-acetyltransferase to increaseefficiency of the system, while also gaining the ability to convertethanol to acetate efficiently.

10) Adding and overexpressing enzymes for the production of CBDA (OS-OACfusion enzyme, CsPti, CBDA Synthase), constructed in a single vector.These enzymes are codon optimized.

11) Grow colonies while adding free fatty acids, and hexanoic acid (forTHCA, CBDA, CBGA, CBCA) or butyric acid (for THCVA, CBDVA, CBGVA,CBCVA).

12) For production of THCA/THCVA, use THCA synthase in step 10 insteadof CBDA synthase. For production of CBGA/CBGVA, follow step 10 but don'tuse CBDA synthase in vector construct. For production of CBCA/CBCVA, useCBC synthase in step 10 instead.

Our strategy for Pichia pastoris (Pichia Pink 1, 2, 3 from Invitrogen)yeast was similar to S. cerevisiae, except for the followingdifferences:

1) Each enzyme, vector, and primer were optimized for insertion intopichia cells instead of S. cerevisiae.

2) Methanol is used to supplement cells in addition to free fatty acid,hexanoic acid, and butyric acid, thereby reducing the total cost ofproduction many fold, while eliminating any contamination issues fromother species.

3) No EMS mutagenesis is performed.

4) Knockouts of pep4 (encoding Proteinase A), prb 1 (encoding ProteinaseB), and YPS1 genes are also introduced. These knockouts allow for theintegration of high copy plasmids leading to higher yields.

5) Steps 7, 8, and 9 from the S. cerevisiae strategy above are not to beperformed in pichia cells.

Example 5 (Cell Free Expression Systems)

Vectors are introduced into cell free expression systems, and makeeither enzymes or intermediate/final products. Further processing orsteps are needed to get purified final products.

Procedures

EMS Mutagenesis (S. cere.; BY4741, BY4742, CEN. PK, CEN. PK2, BY300)

1) Cells incubated overnight @ 30 C in 5 mL TPD medium while shaking @200 rpm to establish 200 mL YPD shake flask culture.

2) When OD600 of yeast culture reaches 1.0, cells are spun down bycentrifugation (12 mins at 4,000 g), washed twice with 20 mL 0.1M sodiumphosphate buffer, pH7.0.

3) Cells concentrated by centrifugation again, re-suspended in 1 mL 0.1Msodium phosphate buffer, transferred to 30 mL FALCON tubes, treated with300 uL EMS (1.2 g/mL).

4) Cells are incubated at 30 C for 1 hr while shaking.

5) Stop mutagenesis by adding 8 mL of sterile 5% sodium thiosulfate toyeast cells.

6) Cells are pelleted, washed with 8 mL sterile water, concentrated bycentrifugation, re-suspended in 1 mL sterile water and 100 uL aliquotsplated into YPD-NCS agar plate (YPD+50 mg/L each of cholesterol,nystatin, sqalestatin, and 2% Bacto-agar).

7) In some instances, washed cells were resuspended in 1 mL YPDE liquidmedia for overnight recovery before plating to YPD-NCS agar medium.

8) Incubate cultures for up to two weeks at 30 C until distinct coloniesare visible.

Bacteria & Yeast Culturing

1) Grown using standard culture practices.

2) YPD media without selection consisted of 1% Bacto-yeast extract, 2%Bacto-peptone, and 2% glucose.

3) Add 40 mg/L ergosterol to YPD media to get YPDE media.

4) Add 40 mg/L each of nystatin, cholesterol, and squalestatin to YPDmedia to get TPDNCS media.

5) Add 40 mg/L each of ergosterol and squalestatin to YPD media to getYPDSE media.

6) Prepare minimal media, SCE (pH5.3), by adding 0.67% Bacto-yeastnitrogen base (without amino acids), 2% dextrose, 0.6% succinic acid,0.14% Sigma yeast dropout soln (-his, -leu, -ura, -trp), uracil (300mg/L), L-tryptophan (150 mg/L), L-histidine (250 mg/L), L-methionine(200 mg/L), L-leucine (1 g/L), and 40 mg/L of ergosterol.

7) Cholesterol and ergosterol stocks are 10 mg/mL in 50% Triton X-100,50% ethanol and kept at −20 C.

8) Selection media prepared similarly except without supplementation ofmedia with indicated reagent based on the yeast auxotrophic markers.

9) All solid media plates are prepared with 2% Bacto-agar. YeastTransformation & Culture Performance

1) Used FROZEN-EZ Yeast Transformation II Kit from Zymo Research,Orange, Calif., according to manufacturer's recommendations.

2) 1 ug of plasmid was used per transformation, followed by selection onagar plates of SCE medium lacking specified amino acids for auxotrophicmarkers, or YPDE containing 300 mg/L hygromycin B for screening erg9knockout at 30 C.

3) Colonies are picked and used to start 3 mL cultures in minimal mediato characterize their terpene production capabilities. (6 daysincubation at 30 C while shaking)

4) Best cultures are chosen to move further, using 30 mL shake flaskcultures.

5) Cultures are grown to saturation in minimal media, inoculated into 30mL SCE media and 1 mL aliquots are taken out daily for 15 days.

6) Cell growth is monitored via change in optical density at 600 nmevery two days using dilutions at later stages of growth.

7) Production of terpenes is determined via testing.

ERG9 Knockout Mutations

1) Primers ERG9PS1 and ERG9-250downS2 used to amplify hygromycinresistance gene, hphNT1, from the pFA6-hph-NT1 vector.

2) Simulataneously add 42 bp nucleotide sequences homologous to regionssurrounding ERG9 gene in yeast genome.

3) Purified PCR fragment is transformed into various cell linesidentified in phase 2 with the ability to accumulate farnesol andselected on YPDE plates containing 300 mg/L hygromycin.

4) Independent single colonies are picked for ergosterol dependent test,PCR confirmation of recombination with hphF and ERG9 450DWR primer.

5) Farnesol production analysis done by GC-MS/LC-MS.

ERG1 Knockout Mutations

1) Primers ERG1F and ERG1R used to amplify the sqalene epoxidasesynthase ERG1 gene by using Takara high fidelity Primerstar taqpolymerase.

2) Obtained PCR fragment is gel purified, A tailed and ligated into thepGEM-Teasy vector.

3) Obtained vector is used as template to run second PCR with primersErg1-splitF and EGR1-splitR to obtain PCR fragment with deletion of 891bp CDS in the middle, yet containing 310 bp at 5′ end region and 291 bpat 3′ end region of ERG1 gene which are the target homologousrecombination sequence for ERG1 knockout.

4) After digestion with BamHI, self-ligation, and transformation toDH5alpha competent cells, resulting vector is pGEM-ERG1-split.

5) Padh-Kanmx4-Tcyc-LoxP antibiotic selection marker cassette isconstructed by assembly PCR of three fragments.

6) Padh promoter is PCR amplified with Padh-loxP-ManHIF and Padh-Kanmx4Rprimers using Yep352 vector as a template.

7) Kanmx4 selection gene is PCR amplified using Padh-kanmx4F andTcyc-kanmx4R primers using PYM-N14 plasmid as a template.

8) Tcyc terminator was PCR amplified with Padh-loxP-BamHIF andPadh-Kanmx4R primers using Pesc vector as a template.

9) 3 PCR fragments containing homologous regions with each other weregel purified and 250 ng of each fragment were mixed together to serve astemplate for the secondary assembly PCR reaction to yieldpAdh-Kanmx4-Tcyc-LoxP cassette.

10) Cassette is digested and inserted into pGEM-ERG1-split vector, andused as template to run PCR with ERG1F and ERG1R to get PCR fragmentused to generate cell lines.

11) Pgpd-tHMGR-Tadh fragment was amplified from Pesc-Gpd-leu-tHMGRvector with primers GPD-BamHIP and Tadh-XholIR.

12) Insert fragment into pGEM-ERG1-split vector containing kanmx4cassette.

13) Use construct as template to amplify with ERG1F and EGR1R primers togain the fragment for building slightly different cell lines, whichinclude integration of one copy of tHMGR into the ERG1 gene.

Primer Name Primer Sequence ERG9pS1TACATTTCATAGCCCATCTTCAACAACAATACCGACTTACCCGTACGCTGCAG (SEQ ID NO: 1)GTCGAC ERG9 250dwS2CAGATTGACGGAGAGAGGGCCACATTGTTTGTCGGCAATAAATCGATGAATTCG (SEQ ID NO: 2)AGCTCG Hph F ATGGGTAAAAAGCCTGAACTCA (SEQ ID NO: 3) Hph RTTATTCCTTTGCCCTCGGACGAG (SEQ ID NO: 4) ERG9 450dwRAGATGCTAGTCAATGGCAGAAG (SEQ ID NO: 5) ERG9p300upF TGCTTACACAGAGTGAACCTGC(SEQ ID NO: 6) ERG9 300R CTCGTGGAAGTGACGCAAC (SEQ ID NO: 7) pGPD-BamHI FcgGGATCCagtttatcattatcaatactcgcc (SEQ ID NO: 8) pGPD-NotIRgggGCGGCCGCgagctcagtdatcattatc (SEQ ID NO: 9) tHMGR-NotIFGGGGCGGCCGCAAAACAATGTTGTCACGACTTTTCCGTATGC (SEQ ID NO: 10) tHMGR-SpeIRGACTAGT TCAAGCTGACTTCTTGGTGCACGTTCCTTG (SEQ ID NO: 11) ERG1FATGTCTGCTGTTAACGTTGCACCTG (SEQ ID NO: 12) ERG1R TTAACCAATCAACTCACCAAAC(SEQ ID NO: 13) ERG1-split F CGGGATCCCTCGAG TTGTTCGCTGCTGACAGCGATAAC(SEQ ID NO: 14) ERG1-splitR CGGGATCCGCTAGCGGTACCACATGGGTCCTTTATATTGACACG(SEQ ID NO: 15) ERG1 90up F ATCAGAACAATTGTCCAGTATTG (SEQ ID NO: 16)ERG1100dwR AATGTACTATACAAGCCTTCC (SEQ ID NO: 17) bSQS-NotIFGGGGCGGCCGCAAAACAATGGGGATGCTTCGCTGGGGAGT (SEQ ID NO: 18) bSQS-SpeIRGACTAGTTTAGCTCCTCAATTCGTCAAAGGT (SEQ ID NO: 19) Cre-NotIFGGGGCGGCCGCAAAACAATGGACATGTTCAGGGATCGCCAGG (SEQ ID NO: 20) Cre-SpeIRGACTAGTCTAATCGCCATCTTCCAGCAGGCG (SEQ ID NO: 21) Padh-Loxp-BamHIFCGGGATCCATAACTTCGTATAGCATACATTATACGAAGTTATGTGGAATATTTC (SEQ ID NO: 22)GGATAT Padh-Kanmx4FGCATACAATCAACTAAGCTAAGCTAAAACAATGGGTAAGGAAAAGACTCACGTT (SEQ ID NO: 23)TC Padh-Kanmx4R GAAACGTGAGTCTTTTCCTTACCCATTGTTTTAGCTTAGCTTAGTTGATTGTAT(SEQ ID NO: 24) GC Kanmx4-TcycFCATTTGATGCTCGATGAGTTTTTCTAAATCCGCTCTAACCGAAAAGGAAGGAG (SEQ ID NO: 25)Kanmx4-TcycR CTCCTTCCTTTTCGGTTAGAGCGGATTTAGAAAAACTCATCGAGCATCAAATG(SEQ ID NO: 26) Tcyc-LoxP-NheIRGGGGCTAGCATAACTTCGTATAATGTATGCTATACGAAGTTATCTTCGAGCGTC (SEQ ID NO: 27)CCAAAA Gpd-BamHIF CGGGATCCAGTTTATCATTATCAATACTCG (SEQ ID NO: 28)Tadh-XhoIR GGGCTCGAGGAGCGACCTCATGCTATCCTG (SEQ ID NO: 29) Kanmx4RTTAGAAAAACTCATCGAGCATC (SEQ ID NO: 30)

Expression of Enzymes for Cannabinoid Production

LS 5 ′FWD SEQ ID NO: 31 Length: 55 Type: DNAOrganism; Artificial Sequence Notes: PrimerGcatagcaatctaatctaagtttaaa atgaatcatttgagagcagaagggcctgc CB 5′ FWDSEQ ID NO: 32 Length: 56 Type: DNA Organism: Artificial SequenceNotes: Primer caccagaacttagtttcgacggataaa atggaaaccggtttgtcctcggtttgcacAll REV SEQ ID NO: 33 Length: 58 Type: DNA Organism: Artificial SequencecataactaattacatgatttaaccTTAAACATCAGATTCAATAGAGCCGCCTCCACTGBackbone |CGBA synthase|Flexible spacer|CBD synthase target peptideSEQ ID NO: 34 Length:  Type: DNA Organism: artiticial sequenceNotes: Codon optimized 1ggttaaatca tgtaattagt tatgtcacgc ttacattcac gccctccccc cacatccgct 61ctaaccgaaa aggaaggagt tagacaacct gaagtctagg tccctattta tttttttata 121gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt tctgtacaga 181cgcgtgtacg catgtaacat tatactgaaa accttgcttg agaaggtttt gggacgctcg 241aaggctttaa tttgcggccc ctcacctgca cgcaaaatag gataattata ctctatttct 301caacaagtaa ttggttgttt ggccgagcgg tctaaggcgc ctgattcaag aaatatcttg 361accgcagtta actgtgggaa tactcaggta tcgtaagatg caagagttcg aatctcttag 421caaccattat ttttttcctc aacataacga gaacacacag gggcgctatc gcacagaatc 481aaattcgatg actggaaatt ttttgttaat ttcagaggtc gcctgacgca tatacctttt 541tcaactgaaa aattgggaga aaaaggaaag gtgagagcgc cggaaccggc ttttcatata 601gaatagagaa gcgttcatga ctaaatgctt gcatcacaat acttgaagtt gacaatatta 661tttaaggacc tattgttttt tccaataggt ggttagcaat cgtcttactt tctaactttt 721cttacctttt acatttcagc aatatatata tatatatttc aaggatatac cattctaatg 781tctgccccta agaagatcgt cgttttgcca ggtgaccacg ttggtcaaga aatcacagcc 841gaagccatta aggttcttaa agctatttct gatgttcgtt ccaatgtcaa gttcgatttc 901gaaaatcatt taattggtgg tgctgctatc gatgctacag gtgttccact tccagatgag 961gcgctggaag cctccaagaa ggctgatgcc gttttgttag gtgctgtggg tggtcctaaa 1021tggggtaccg gtagtgttag acctgaacaa ggtttactaa aaatccgtaa agaacttcaa 1081ttgtacgcca acttaagacc atgtaacttt gcatccgact ctcttttaga cttatctcca 1141atcaagccac aatttgctaa aggtactgac ttcgttgttg tcagagaatt agtgggaggt 1201atttactttg gtaagagaaa ggaagatgat ggtgatggtg tcgcttggga tagtgaacaa 1261tacaccgttc cagaagtgca aagaatcaca agaatggccg ctttcatggc cctacaacat 1321gagccaccat tgcctatttg gtccttggat aaagctaatg ttttggcctc ttcaagatta 1381tggagaaaaa ctgtggagga aaccatcaag aacgaattcc ctacattgaa ggttcaacat 1441caattgattg attctgccgc catgatccta gttaagaacc caacccacct aaatggtatt 1501ataatcacca gcaacatgtt tggtgatatc atctccgatg aagcctccgt tatcccaggt 1561tccttgggtt tgttgccatc tgcgtccttg gcctctttgc cagacaagaa caccgcattt 1621ggtttgtacg aaccatgcca cggttctgct ccagatttgc caaagaataa ggtcaaccct 1681atcgccacta tcttgtctgc tgcaatgatg ttgaaattgt cattgaactt gcctgaagaa 1741ggtaaggcca ttgaagatgc agttaaaaag gttttggatg caggcatcag aactggtgat 1801ttaggtggtt ccaacagtac caccgaagtc ggtgatgctg tcgccgaaga agttaagaaa 1861atccttgctt aaaaagattc tcttttttta tgatatttgt acataaactt tataaatgaa 1921attcataata gaaacgacac gaaattacaa aatggaatat gttcataggg taacgctatg 1981atccaatatc aaaggaaatg atagcattga aggatgagac taatccaatt gaggagtggc 2041agcatataga acagctaaag ggtagtgctg aaggaagcat acgatacccc gcatggaatg 2101ggataatatc acaggaggta ctagactacc tttcatccta cataaataga cgcatataag 2161tacgcattta agcataaaca cgcactatgc cgttcttctc atgtatatat atatacaggc 2221aacacgcaga tataggtgcg acgtgaacag tgagctgtat gtgcgcagct cgcgttgcat 2281tttcggaagc gctcgttttc ggaaacgctt tgaagttcct attccgaagt tcctattctc 2341tagaaagtat aggaacttca gagcgctttt gaaaaccaaa agcgctctga agtcgcactt 2401tcaaaaaacc aaaaacgcac cggactgtaa cgagctacta aaatattgcg aataccgctt 2461ccacaaacat tgctcaaaag tatctctttg ctatatatct ctgtgctata tccctatata 2521acctacccat ccacctttcg ctccttgaac ttgcatctaa actcgacctc tacatttttt 2581atgtttatct ctagtattac tctttagaca aaaaaattgt agtaagaact attcatagag 2641tgaatcgaaa acaatacgaa aatgtaaaca tttcctatac gtagtatata gagacaaaat 2701agaagaaacc gttcataatt ttctgaccaa tgaagaatca tcaacgctat cactttctgt 2761tcacaaagta tgcgcaatcc acatcggtat agaatataat cggggatgcc tttatcttga 2821aaaaatgcac ccgcagcttc gctagtaatc agtaaacgcg ggaagtggag tcaggctttt 2881tttatggaag agaaaataga caccaaagta gccttcttct aaccttaacg gacctacagt 2941gcaaaaagtt atcaagagac tgcattatag agcgcacaaa ggagaaaaaa agtaatctaa 3001gatgctttgt tagaaaaata gcgctctcgg gatgcatttt tgtagaacaa aaaagaagta 3061tagattcttt gttggtaaaa tagcgctctc gcgttgcatt tctgttctgt aaaaatgcag 3121ctcagattct ttgtttgaaa aattagcgct ctcgcgttgc atttttgttt tacaaaaatg 3181aagcacagat tcttcgttgg taaaatagcg ctttcgcgtt gcatttctgt tctgtaaaaa 3241tgcagctcag attctttgtt tgaaaaatta gcgctctcgc gttgcatttt tgttctacaa 3301aatgaagcac agatgcttcg ttcaggtggc acttttcggg gaaatgtgcg cggaacccct 3361atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 3421tattggtcag aattggttaa ttggttgtaa cactgacccc tatttgttta tttttctaaa 3481tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 3541gaaaaaggaa gaatatgagc catattcaac gggaaacgtc gaggccgcga ttaaattcca 3601acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg 3661cgacaatcta tcgcttgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca 3721aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat 3781ttatgccact tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca 3841ccactgcgat ccccggaaaa acagcgttcc aggtattaga agaatatcct gattcaggtg 3901aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcactcgatt cctgtttgta 3961attgtccttt taacagcgat cgcgtatttc gcctcgctca ggcgcaatca cgaatgaata 4021acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag 4081tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg 4141atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg 4201gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg 4261agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata 4321tgaataaatt gcaatttcat ttgatgctcg atgagttttt ctaactcatg accaaaatcc 4381cttaacgtga gttacgcgcg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa 4441aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 4501accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 4561aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagc 4621ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 4681agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 4741accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 4801gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 4861tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 4921cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 4981cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 5041cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 5101ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 5161taccgctcgg ggtcgtgcag gtagtttatc attatcaata ctcgccattt caaagaatac 5221gtaaataatt aatagtagtg attttcctaa ctttatttag tcaaaaaatt agccttttaa 5281ttctgctgta acccgtacat gcccaaaata gggggcgggt tacacagaat atataacatc 5341gtaggtgtct gggtgaacag tttattcctg gcatccacta aatataatgg agcccgcttt 5401ttaagctggc atccagaaaa aaaaagaatc ccagcaccaa aatattgttt tcttcaccaa 5461ccatcagttc ataggtccat tctcttagcg caactacaga gaacaggggc acaaacaggc 5521aaaaaacggg cacaacctca atggagtgat gcaaccagcc tggagtaaat gatgacacaa 5561ggcaattgac ccacgcatgt atctatctca ttttcttaca ccttctatta ccttctgctc 5641tctctgattt ggaaaaagct gaaaaaaaag gttgaaacca gttccctgaa attattcccc 5701tacttgacta ataagtatat aaagacggta ggtattgatt gtaattctgt aaatctattt 5761cttaaacttc ttaaattcta cttttatagt tagtcttttt tttagtttta aaacaccaga 5821acttagtttc gacggataaa atggaaaccg gtttgtcctc ggtttgcact ttctccttcc 5881aaacaaacta tcatacactc ctgaacccgc acaataacaa tcccaaaact tccctgctgt 5941gttataggca cccaaagaca ccaatcaaat actcctacaa taactttcca tctaagcatt 6001gtagcacaaa aagtttccat ttgcaaaata agtgttccga atctctgtcc atcgccaaaa 6061attccattag ggctgccact actaatcaaa ctgaaccacc agagtctgat aatcattctg 6121tcgccacaaa gattctgaat tttgggaagg cttgttggaa gttacaaaga ccatatacaa 6161ttattgcctt tacctcttgt gcctgtggtt tatttggtaa ggaactgttg cataatacaa 6241atttaatatc ttggtcattg atggaaacgt tcaaagcatt ttttttctta gtcgctatcc 6301tttgtattgc ttctttcacc accactatca accagattta cgacttacat attgacagaa 6361ttaacaagcc agatttgcca ctggcttcgg gcgagatttc cgtcaatact gcctggatca 6421tggaaacttc tattattgtt gccttgtttg gartgataat caccataaaa atggaaacta 6481agggtggtcc attgtatatt ttcggttact gttttggtat cttcgggggc atcgtctact 6541ctgttcctcc attcagatgg aaacaaaatc cttccacagc attccttttg aacttcctgg 6601cgcacattat aaccaacttt actttttatt atgcctccag agccgccctg gggctgccct 6661ttgaattacg cccctccttt acatttttac tggccttcat ggagaccaag tccatggaga 6721ctggttctgc tctcgcgttg atcaaagatg cttccgatgt ggaaggtgac accaaatttg 6761gtatatccac tttggccagc aagtatggtt ccaggaattt gaccctattt tgttctggta 6841tcgtgctgct gtcttatgtt gcagccatct tggctggcat catttggcca caggctttca 6901attcaaatgt tatggagacg ctgctctcgc atgctatttt ggcattttgg ttgattctac 6961agacaagaga ttttgcttta accaattatg acccagaagc tggtagaaga ttttacgaat 7021ttatggaaac atggaaatta tactatgctg aatatttagt gtacgttttc attgggggcg 7081gctccagcgc cggcggcggc tcttctgcgg gcggttggtc tcatccacaa tttgagaaag 7141gtgggtcgtc tggcggcggc agcgggggcg ggtccggcgg ggggagcggc ggtatgaaat 7201gttcgacctt ctctttttgg tttgtctgta aaataatttt ttttttcttc agctttaaca 7261ttcaaaccag cattgcaaat ccaagagaaa atttcttgaa atgcttttca caatatatcc 7321ccaataatgc tactaacttg aagctagttt atactcaaaa caaccctttg tacatgtccg 7361tgctcaactc caccattcac aacctaagat tcacttcaga cactacccca aaaccattag 7441ttattgtgac accttctcac gtttcacata tccaaggtac tattttatgc tccaagaagg 7501tcggcctgca aattagaact agatctggag gtcatgattc agaaggaatg tcttacatct 7561ctcaagttcc atttgtgatt gtcgatttaa gaaatatgag gagcattaag atcgatgttc 7621actcccaaac ggcatgggtt gaagccggtg ccaccttggg cgaagtttac tactgggtca 7681acgagaagaa tgaaaactta tcactagccg caggttattg tccaactgtt tgtgctggtg 7741gccatttcgg aggcggcggc tacggtcctc taatgagaaa ctacggctta gctgctgaca 7801atatcatcga cgctcacttg gttaacgttc atggtaaagt tttagataga aaatctatgg 7861gtgaggatct tttctgggct ttgagaggtg gcggcgcaga atcatttggc attatcgttg 7921cttggaagat cagattggtg gctgtcccca agtctacaat gttttctgtg aagaaaatta 7961tggaaatcca tgaattggtc aaactggtga ataaatggca aaacatagct tacaagtacg 8041ataaagactt gctgttaatg acacatttta ttaccaggaa catcactgat aaccaaggca 8101agaacaagac tgcaattcat acttattttt cctccgtttt tttgggtggt gtcgactccc 8161tcgtggatct gatgaataaa tcattccctg aactaggtat taaaaaaacc gattgtagac 8221aattgagttg gattgatacc atcatattct acagtggtgt tgttaattat gatactgaca 8281acttcaacaa agaaatactg ctggaccgtt ccgccggcca gaatggtgct tttaaaatca 8341agttggatta tgtgaaaaag cctattccag aatccgtatt tgttcaaata ttggaaaagc 8401tgtatgaaga agacattggt gcaggcatgt acgctcttta tccttatggc ggcataatgg 8461atgaaatttc tgaaagtgcc attcctttcc cacatagggc cgggatcctg tacgagttat 8521ggtacatttg ttcatgggaa aagcaagaag ataatgaaaa acatttaaat tggataagaa 8561atatttataa ttttatgact ccatacgtct ccaaaaaccc acgcctggca tatttgaatt 8641acagagacct ggatattggc atcaatgatc ctaaaaaccc aaataattac actcaggcaa 8701gaatatgggg tgaaaaatat ttcggcaaaa attttgatag gctggtcaag gttaaaacac 8761tggttgatcc aaacaatttc tttagaaacg aacaatctat cccacctctg cctagacata 8821gacacggcgg tggaagcagt ggaggcggct ctattgaatc tgatgtttaa tgaBackbone |OLS|Flexible spacer|OAC|target peptide SEQ ID NO: 35 Length:Type: DNA Organism: artificial sequence Notes: Codon optimized 1ggttaaatca tgtaattagt tatgtcacgc ttacattcac gccctccccc cacatccgct 61ctaaccgaaa aggaaggagt tagacaacct gaagtctagg tccctattta tttttttata 121gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt tctgtacaga 181cgcgtgtacg catgtaacat tatactgaaa accttgcttg agaaggtttt gggacgctcg 241aaggctttaa tttgcggccc ctcacctgca cgcaaaaagc ttttcaattc aattcatcat 301ttttttttta ttcttttttt tgatttcggt ttctttgaaa tttttttgat tcggtaatct 361ccgaacagaa ggaagaacga aggaaggagc acagacttag attggtatat atacgcatat 421gtagtgttga agaaacatga aattgcccag tattcttaac ccaactgcac agaacaaaaa 481ccagcaggaa acgaagataa atcatgtcga aagctacata taaggaacgt gctgctactc 541atcctagtcc tgttgctgcc aagctattta atatcatgca cgaaaagcaa acaaacttgt 601gtgcttcatt ggatgttcgt accaccaagg aattactgga gttagttgaa gcattaggtc 661ccaaaatttg tttactaaaa acacatgtgg atatcttgac tgatttttcc atggagggca 721cagttaagcc gctaaaggca ttatccgcca agtacaattt tttactcttc gaagatagaa 781aatttgctga cattggtaat acagtcaaat tgcagtactc tgcgggtgta tacagaatag 841cagaatgggc agacattacg aatgcacacg gtgtggtggg cccaggtatt gttagcggtt 901tgaagcaggc ggcagaagaa gtaacaaagg aacctagagg ccttttgatg ttagcagaat 961tgtcatgcaa gggctcccta tctactggag aatatactaa gggtactgtt gacattgcga 1021aaagcgacaa agattttgtt atcggcttta ttgctcaaag agacatgggt ggaagagatg 1081aaggttacga ttggttgatt atgacacccg gtgtgggttt agatgacaag ggagatgcat 1141tgggtcaaca gtatagaacc gtggatgatg ttgtctctac aggatctgac attattattg 1201ttggaagagg actatttgca aagggaaggg atgctaaggt agagggtgaa cgttacagaa 2161aagcaggctg ggaagcatat ttgagaagat gcggccagca aaactaaaaa actgtattat 1321aagtaaatgc atgtatacta aactcacaaa ttagagcttc aatttaatta tatcagttat 1381tacccacgct atgatccaat atcaaaggaa atgatagcat tgaaggatga gactaatcca 1441attgaggagt ggcagcatat agaacagcta aagggtagtg ctgaaggaag catacgatac 1501cccgcatgga atgggataat atcacaggag gtactagact acctttcatc ctacataaat 1561agacgcatat aagtacgcat ttaagcataa acacgcacta tgccgttctt ctcatgtata 1621tatatataca ggcaacacgc agatataggt gcgacgtgaa cagtgagctg tatgtgcgca 1681gctcgcgttg cattttcgga agcgctcgtt ttcggaaacg ctttgaagtt cctattccga 1741agttcctatt ctctagaaag tataggaact tcagagcgct tttgaaaacc aaaagcgctc 1801tgaagtcgca ctttcaaaaa accaaaaacg caccggactg taacgagcta ctaaaatatt 1861gcgaataccg cttccacaaa cattgctcaa aagtatctct ttgctatata tctctgtgct 1921atatccctat ataacctacc catccacctt tcgctccttg aacttgcatc taaactcgac 1981ctctacattt tttatgttta tctctagtat tactctttag acaaaaaaat tgtagtaaga 2041actattcata gagtgaatcg aaaacaatac gaaaatgtaa acatttccta tacgtagtat 2101atagagacaa aatagaagaa accgttcata attttctgac caatgaagaa tcatcaacgc 2161tatcactttc tgttcacaaa gtatgcgcaa tccacatcgg tatagaatat aatcggggat 2221gcctttatct tgaaaaaatg cacccgcagc ttcgctagta atcagtaaac gcgggaagtg 2281gagtcaggct ttttttatgg aagagaaaat agacaccaaa gtagccttct tctaacctta 2341acggacctac agtgcaaaaa gttatcaaga gactgcatta tagagcgcac aaaggagaaa 2401aaaagtaatc taagatgctt tgttagaaaa atagcgctct cgggatgcat ttttgtagaa 2461caaaaaagaa gtatagattc tttgttggta aaatagcgct ctcgcgttgc atttctgttc 2521tgtaaaaatg cagctcagat tctttgtttg aaaaattagc gctctcgcgt tgcatttttg 2581ttttacaaaa atgaagcaca gattcttcgt tggtaaaata gcgctttcgc gttgcatttc 2641tgttctgtaa aaatgcagct cagattcttt gtttgaaaaa ttagcgctct cgcgttgcat 2701ttttgttcta caaaatgaag cacagatgct tcgttcaggt ggcacttttc ggggaaatgt 2761gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 2821acaataaccc tgatattggt cagaattggt taattggttg taacactgac ccctatttgt 2881ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg 2941cttcaataat attgaaaaag gaagaatatg agtattcaac atttccgtgt cgcccttatt 3001cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta 3061aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc 3121ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa 3181gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc 3241cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt 3301acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact 3361gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac 3421aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata 3481ccaaacgacg agcgtgacac cacgatgcct gtagcgatgg caacaacgtt gcgcaaacta 3541ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg 3601gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat 3661aaatccggag ccggtgagcg tggttctcgc ggtatcatcg cagcgctggg gccagatggt 3721aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga 3781aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact catgaccaaa 3841atcccttaac gtgagttacg cgcgcgtcgt tccactgagc gtcagacccc gtagaaaaga 3901tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 3961aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 4021aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt 4081tagcccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 4141taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 4201agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 4261tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 4321cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 4381agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 4441gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 4501aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 4561tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 4621ctgataccgc tcggggtcgt gcaggtatag cttcaaaatg tttctactcc ttttttactc 4681ttccagattt tctcggactc cgcgcatcgc cgtaccactt caaaacaccc aagcacagca 4741tactaaattt cccctctttc ttcctctagg gtgtcgttaa ttacccgtac taaaggtttg 4801gaaaagaaaa aagtgaccgc ctcgtttctt tttcttcgtc gaaaaaggca ataaaaattt 4861ttatcacgtt tctttttctt gaaaattttt ttttttgatt tttttctctt tcgatgacct 4921cccattgata tttaagttaa taaacggact tcaatttctc aagtttcagt ttcatttttc 4981ttgttctatt acaacttttt ttacttcttg ctcattagaa agaaagcata gcaatctaat 5041ctaagtttaa aatgaatcat ttgagagcag aagggcctgc ttccgtgctg gctattggta 5101ccgccaatcc agaaaatatc ctgctgcagg acgaattccc agattactat tttagggtca 5161ccaaatctga acatatgaca caattgaaag agaaattcag aaagatttgt gacaagtcca 5221tgattaggaa aagaaattgt tttttgaatg aagaacactt gaagcaaaat cctcgcctgg 5281tggagcatga aatgcaaact ttggatgcta gacaagacat gttggtggtg gaagttccaa 5341agctggggaa ggatgcctgt gccaaggcca ttaaagaatg gggccaacca aaatccaaaa 5401ttacccacct gattttcacc tccgcctcca ccactgatat gccaggtgca gactatcatt 5461gtgctaaatt gttgggtttg tccccctccg tgaagagagt tatgatgtat caattaggtt 5521gttatggcgg cggcaccgtt ctgagaattg ccaaagacat tgctgaaaac aataaaggtg 5581cgcgcgtttt ggctgtttgt tgtgatatta tggcatgttt atttagaggt ccaagtgaaa 5641gtgacttgga attgctagtg ggccaggcca tatttggtga tggtgccgct gctgtgatcg 5701ttggtgctga gcctgatgaa tctgtcggtg aaagaccaat ttttgaactg gtttccactg 5761gtcaaaccat tttgccaaat tcagaaggta ctattggcgg ccatatcaga gaagctggtt 5821taatctttga tttgcacaag gatgtcccaa tgttaatttc caataatatt gaaaaatgtt 5881tgatcgaagc atttaccccc atcggtattt ctgattggaa ttccatcttc tggattacac 5941atcctggcgg taaagctatc ttagataaag ttgaggagaa gttgcattta aagtctgaca 6001aatttgttga ttcaagacat gtcctgtctg agcacggtaa tatgtcttcc tcgaccgtct 6061tgtttgtcat ggatgagttg aggaagaggt ccctggaaga aggcaagagc accaccggtg 6121acggttttga gtggggggtc ctctttggat ttgggccagg cctgaccgta gaaagggttg 6181ttgtccgctc ggtgccaatc aaatatggtg gggggtccag cgccggtggc gggagctccg 6241cgggcggttg gtctcaccca caatttgaaa agggtggcag cagcggcggc ggctctggcg 6301gaggctccgg cgggggctcg gggggtatgg ctgtcaagca tctgatcgtg ctgaagttca 6361aagatgaaat tactgaagcc caaaaggagg aatttttcaa gacatatgtt aatttggtta 6421acatcattcc agcaatgaaa gatgtttatt ggggtaagga cgttactcaa aaaaataagg 6481aagagggtta cactcatatt gttgaagtca ctttcgaatc cgtcgaaaca attcaagatt 6541atattattca tccagctcat gttgggtttg gcgatgtgta cagatcattt tgggaaaaat 6601tattgatttt tgactacaca ccaagaaaag gcggtggaag cagtggaggc ggctctattg 6661aatctgatgt ttaatagOverexpression of ERG8m HFA1, ERG 10, ERG13, tHMGR, HMGR,ERG 12, ERG8, IDI Genes (for higher levels of intermediates)Same process as expression of Synthase expression, but with 3copies expressed in yeast cells.Backbone |GGPS1|2a protease |HMC-CoA rcductase|flexible spacer IDI1SEQ ID NO: 36 Length: Type: DNA Organism: artificial sequenceNotes: Codon optimized 1atggagaaga ctcaagaaac agtccaaaga attcttctag aaccctataa atacttactt 61cagttaccag gtaaacaagt gagaaccaaa ctttcacagg catttaatca ttggctgaaa 121gttccagagg acaagctaca gattattatt gaagtgacag aaatgttgca taatgccagt 181ttactcatcg atgatattga agacaactca aaactccgac gtggctttcc agtggcccac 241agcatctatg gaatcccatc tgtcatcaat tctgccaatt acgtgtattt ccttggcttg 301gagaaagtct taacccttga tcacccagat gcagtgaagc tttttacccg ccagcttttg 361gaactccatc agggacaagg cctagatatt tactggaggg ataattacac ttgtcccact 421gaagaagaat ataaagctat ggtgctgcag aaaacaggtg gactgtttgg attagcagta 481ggtctcatgc agttgttctc tgattacaaa gaagatttaa aaccgctact taatacactt 541gggctctttt tccaaattag ggatgattat gctaatctac actccaaaga atatagtgaa 601aacaaaagtt tttgtgaaga tctgacagag ggaaagttct catttcctac tattcatgct 661atttggtcaa ggcctgaaag cacccaggtg cagaatatct tgcgccagag aacagaaaac 721atagatataa aaaaatactg tgtacattat cttgaggatg taggttcttt tgaatacact 781cgtaataccc ttaaagagct tgaagctaaa gcctataaac agattgatgc acgtggtggg 841aaccctgagc tagtagcctt agtaaaacac ttaagtaaga tgttcaaaga agaaaatgaa 901ggcggttctg gcagcggaga gggcagagga agtcttctaa catgcggtga cgtggaggag 961aatcccggcc ctaggtctgg cagcggagag ggcagaggaa gtcttctaac atgcggtgac 1021gtggaggaga atcccggccc taggacacaa aagaaagtcc cagacaattg ttgtagacgt 1081gaacctatgc tggtcagaaa taaccagaaa tgtgattcag tagaggaaga gacagggata 1141aaccgagaaa gaaaagttga ggttataaaa cccttagtgg ctgaaacaga taccccaaac 1201agagctacat ttgtggttgg taactcctcc ttactcgata cttcatcagt actggtgaca 1261caggaacctg aaattgaact tcccagggaa cctcggccta atgaagaatg tctacagata 1321cttgggaatg cagagaaagg tgcaaaattc cttagtgatg ctgagatcat ccagttagtc 1381aatgctaagc atatcccagc ctacaagttg gaaactctga tggaaactca tgagcgtggt 1441gtatctattc gccgacagtt acrttccaag aagctttcag aaccttcttc tctccagtac 1501ctaccttaca gggattataa ttactccttg gtgatgggag cttgttgtga gaatgttatt 1561ggatatatgc ccatccctgt tggagtggca ggaccccttt gcttagatga aaaagaattt 1621caggttccaa tggcaacaac agaaggttgt cttgtggcca gcaccaatag aggctgcaga 1681gcaataggtc ttggtggagg tgccagcagc cgagtccttg cagatgggat gactcgtggc 1741ccagttgtgc gtcttccacg tgcttgtgac tctgcagaag tgaaagcctg gctcgaaaca 1801tctgaagggt tcgcagtgat aaaggaggca tttgacagca ctagcagatt tgcacgtcta 1861cagaaacttc atacaagtat agctggacgc aacctttata tccgtttcca gtccaggtca 1921ggggatgcca tggggatgaa catgatttca aagggtacag agaaagcact ttcaaaactt 1981cacgagtatt tccctgaaat gcagattcta gccgttagtg gtaactattg tactgacaag 2041aaacctgctg ctataaattg gatagaggga agaggaaaat ctgttgtttg tgaagctgtc 2101attccagcca aggttgtcag agaagtatta aagactacca cagaggctat gattgaggtc 2161aacattaaca agaatttagt gggctctgcc atggctggga gcataggagg ctacaacgcc 2221catgcagcaa acattgtcac cgccatctac attgcctgtg gacaggatgc agcacagaat 2281gttggtagtt caaactgtat tactttaatg gaagcaagtg gtcccacaaa tgaagattta 2341tatatcagct gcaccatgcc atctatagag ataggaacgg tgggtggtgg gaccaaccta 2401ctacctcagc aagcctgttt gcagatgcta ggtgttcaag gagcatgcaa agataatcct 2461ggggaaaatg cccggcagct tgcccgaatt gtgtgtggga ccgtaatggc tggggaattg 2521tcacttatgg cagcattggc agcaggacat cttgtcaaaa gtcacatgat tcacaacagg 2581tcgaagatca atttacaaga cctccaagga gcttgcacca agaagacagc cggctcagga 2641ggttcttcag gactggaagt gctgtttcag ggcccgggtg gatctggcat gatgcctgaa 2701ataaacacta accacctcga caagcaacag gttcaactcc tggcagagat gtgtatcctt 2761attgatgaaa atgacaataa aattggagct gagaccaaga agaattgtca cctgaacgag 2821aacattgaga aaggattatt gcatcgagct tttagtgtct tcttattcaa caccgaaaat 2881aagcttctgc tacagcaaag atcagatgct aagattacct ttccaggttg ttttacgaat 2941acgtgttgta gtcatccatt aagcaatcca gccgagcttg aggaaagtga cgcccttgga 3001gtgaggcgag cagcacagag acggctgaaa gctgagctag gaattccctt ggaagaggtt 3061cctccagaag aaattaatta tttaacacga attcactaca aagctcagtc tgatggtatc 3121tggggtgaac atgaaattga ttacattttg ttggtgagga agaatgtaac tttgaatcca 3181gatcccaatg agattaaaag ctattgttat gtgtcaaagg aagaactaaa agaacttctg 3241aaaaaagcag ccagtggtga aattaagata acgccatggt ttaaaattat tgcagcgact 3301tttctcttta aatggtggga taacttaaat catttgaatc agtttgttga ccatgagaaa 3361atatacagaa tg

TABLE 1 Compounds Pharmacological Characteristics Cannabinoids (FIG. 1and 2) Cannabigerolic acid (CBGA) Antibiotic (1) Cannabigerolic acidmonomethylether (CBGAM) Cannabigerol (CBG) Antibiotic, antifungal,anti-inflammatory, analgesic (1) Partial agonist at CB1/CB2 receptors(2) Cannabigerovarinic acid (CBGVA) Cannabigerovarin (CBGV)Cannabichromenic acid (CBCA) Cannabichromene (CBC) Anti-inflammatory,antibiotic, antifungal, analgesic (1) Cannabichromevarinic acid (CBCVA)Cannabichromevarin (CBCV) Cannabidiolic acid (CBDA) AntibioticCannabidiol (CBD) Anxiolytic, antipsychotic, analgesic,anti-inflammatory, antioxidant, antispasmodic (1) Ant schizophrenic,antiepileptic, sleep-promoting, anti-oxidizing, anti-inflammatory,immunomodulation properties (2) Cannabidiol monomethylether (CBDM)Cannabidiol-C4 (CBD-C4) Cannabidivarinic acid (CBDVA) Cannabidivarin(CBDV) Cannabidiorcol (CBD-C1) Tetrahydrocannabinolic acid A (THCA-A)Tetrahydrocannabinolic acid B (THCA-B) Delta-9-tetrahydrocannabinolEuphoriant, analgesic, anti- (THC) inflammatory, antioxidant, antiemetic(1) Delta-9-tetrahydrocannabinolic acid-C4 (THCA-C4)Delta-9-tetrahydrocannabinol-C4 (THC-C4) Delta-8-tetrahydrocannabivarinExhibit in vitro pharma (D8-THCV) properties similar to THCV, and bothcan antagonize THC; behave as agonists or antagonists in dose dependentmanner (2) Delta-9-tetrahydrocannabivarinic acid (THCVA)Delta-9-tetrahydrocannabivarin Analgesic, euphoriant (1) (THCV) Strongantagonist of anandamide (due to interactions with non- CB1/2receptors), neuromodulator (in animal and human organs), some affectsdue to interaction with non CB1/CB2 receptors (2)Delta-9-tetrahydrocannabiorcolic acid (THCA-C1)Delta-9-tetrahydrocannabiorcol (THC-C1) Delta-7-cis-iso-tetrahydrocannabivarin (D7-THCV) Delta-8- tetrahydrocannabinolic acid(D8-THCA) Delta-8-tetrahydrocannabinol Similar to THC (1) (D8-THC)Several 1-O-methyl- and 1-deoxy-delta-8- THC analogs have high CB2receptor affinity[JWH133, JWH359, trans- (6aR,10aR)-3-(1,1-dimethylhexyl)-1-O- methyl-delta-8-THC]; antiemetic effects similar toTHC (2) Cannabicyclolic acid (CBLA) Cannabicyclol (CBL)Cannabicyclovarin (CBLV) Cannabielsoic acid A (CBEA-A) Cannabielsoicacid B (CBEA-B) Cannabielsoin (CBE) Cannabinolic acid (CBNA) Cannabinol(CBN) Sedative, antibiotic, anticonvulsant, anti- inflammatory (1)Cannabinol methylether (CBNM) Cannabinol-C4 (CBN-C4) Cannabivarin (CBV)Cannabinol-C2 (CBN-C2) Cannabinol-C1 (CBN-C1) Cannabinodiol (CBND)Cannabinodivarin (CBVD) Cannabitriol (CBT) 10-Ethoxy-9-hydroxy-delta-6a-tetrahydrocannabinol 8,9-Dihydroxy-delta-6a-tetrahydrocannabinol Cannabitriolvarin (CBTV) Ethoxy-cannabitriolvarin(CBTVE) Dehydrocannabifuran (DCBF) Cannabifuran (CBF) Cannabichromanon(CBCN) Cannabicitran (CBT) 10-oxo-delta-6a- tetrahydrocannabinol (OTHC)Delta-9-cis- tetrahydrocannabinol (Cis-THC)3,4,5,6-Tetrahydro-7-hydroxy- alpha-alpha-2-trimethyl-9-n-propyl-2,6-methano-2H-1- benzoxocin-5-methanol (OH-iso-HHCV)Cannabiripsol (CBR) Trihydroxy-delta-9- tetrahydrocannabinol (triOH-THC)Terpeses/Terpenoids Beta-Myrcene Analgesic, anti-inflammatory,antibiotic, antimutagenic d-Limonene Immune potentiator, antidepressant,antimutagenic Linalool Sedative, antidepressant, anxiolytic, immunepotentiator Trans-Ocimene Beta-Pinene Alpha-Pinene Anti-inflammatory,bronchodilator, stimulant, antibiotic, antineoplastic, AChE inhibitorBeta-Caryophyllene Anti-inflammatory, cytoprotective, antimalarial, CB2agonist Delta-3-Carene Pulegone AChE inhibitor, sedative,Trans-gamma-Bisabolene antipyretic Trans-alpha-Farnesene Beta-FencholBeta-Phellandrene Alpha-Humulene Guajol Alpha-Gualene Alpha-EudesmolTerpinolene Alpha-Selinene Alpha-Terpineol Sedative, antibiotic, AChEinhibitor, antioxidant, antimalarial Fenchone Camphene Cis-Sabinenehydrate Cis-Ocimene Beta-Eudesmol Beta-Selinene Alpha-trans-BergamoleneGamma-Eudesmol Borneol Cis-beta-Farnescene Gamma-CurcumeneCis-gamma-Bisabolene Alpha-Thujene Epi-alpha-Bisabolol IpsdienolAlpha-Yiangene Beta-Elemene Alpha-cis-Bergamontene Gamma-MuuroleneAlpha-Cadinene Alpha-Longipinene Caryophyllene oxide SpermidineAlkaloids (FIG. 6) (+)-Cannabisativine Palustridine PalustrineSpermidine Anhydrocannabisativine Phenolic Amides and Lignanamides (FIG.5) N-trans-Feruloyltyramine N-p-CoumaroyltyramineN-trans-Caffeoyltyramine Grossamide Cannabisin-A Cannabisin-BCannabisin-C Cannabisin-D Cannabisin-E Cannabisin-F Cannabisin-GPhenylpropanoids and Flavonoids (FIG. 4) Apigenin Luteolin KaempferolQuercetin Orientin Vitexin Cannflavin A Inhibit prostaglandin E2 inhuman rheumatoid synovial cells Cannflavin B Inhibit prostaglandin E2 inhuman rheumatoid synovial cells Stilbenoids (FIG. 3) CannabispiranIsocannabispiran Cannabistilbene-IIa Cannabistilbene-IIb Cannithrene-1Cannithrene-2 Acetyl cannabispirol Alpha-cannabisporanol CannipreneCannabispirone

TABLE 2 (Starting Materials) Sugar based concentrates (HighHemicellulose Glycerol Fructose Corn Syrup, Molasses) Glucose XyloseWhey Sucrose Methanol Biodiesel Cellulose Lactic Acid Citrate EthanolLignin Fructose Succinic Acid Arabinose Biofuels Biomass SaccharoseStarch based products Agricultural residue Water hyacinth Aquaticbiomass

What is claimed is:
 1. A method for producing enzymes and products,wherein the method comprises: inserting a gene into an expression systemand thereby yielding a modified host; optimizing the modified host andthereby yielding the enzymes and the products; purifying the enzymes andproducts; and wherein the gene contains a codon-optimized nucleic acidsequence of SEQ ID NO: 34 or a nucleic acid sequence that is 95% ormore, 96% or more, 97% or more, 98% or more, or 99% or more homologousto the nucleic acid sequence of SEQ ID NO: 34; or an codon-optimizednucleic acid sequence of nucleic acids 5052 to 6671 of SEQ ID NO: 34 ora nucleotide sequence that is 95% or more, 96% or more, 97% or more, 98%or more, or 99% or more homologous to the nucleic acids 5052 to 6671 ofSEQ ID NO:
 34. 2. The method of claim 1, wherein the products are foundin a Cannabis plant.
 3. The method of claim 1, wherein the productscomprise at least one of: cannabinoids, terpenoids, stilbenoids,flavonoids, phenolic amides, lignanamides, spermidine alkaloids, andphenylpropanoids.
 4. The method of claim 1, wherein the expressionsystem comprises at least one of: a vector, a cosmid, BAC, YAC, and aphage.
 5. The method of claim 1, wherein optimizing the modified hostcomprises: manipulating genes in the the modified host; silencing genesin the modified host; and amplifying genes in the modified host.
 6. Themethod of claim 1, wherein the modified host is a bacteria yeast, plant,alga, or fungus.
 7. The method of claim 5, further comprises:controlling carbon flux in the modified host to yield a product ofinterest.
 8. The method of claim 1, wherein the modified host is S.cerevisiae, E. coli, P. pastorisis, N. crassa, S. pombe, R. palmatum, C.longa, O. sativa, A. arborescens, A. terrus, C. sativa, S. griseus, S.erythera, S. coelicolor, S. toxytruini, S. cellulosum, P. floresceus, A.orientalis, S. livedans, A. vinclandii, B. subtilis, M tuberculosis, M.xanthus, A. oryzae, A. niger, R. palmatum, H. serrata, R. rubra, L.erythrhizon, L. acetobutylicum, C. utilis, A. niger, C. glutamicum, or aspecies of bacillus.
 9. The method of claim 1, further comprisingsupplementing cells with methanol to free fatty acids, hexonoic acid,and butyric acid.
 10. The method of claim 1, wherein the product derivesfrom a sugar molecule.
 11. The method of claim 7, wherein controllingthe carbon flux comprises: uptaking exogenous sterol; and increasingcell permeability.
 12. A method for producing enzymes and products,wherein the method comprises: inserting a gene into an expression systemand thereby yielding a modified host, wherein the expression system isfree of cells; optimizing the modified host and thereby yielding theenzymes and the products; purifying the products; and wherein the genecontains a codon-optimized nucleic acid sequence of SEQ ID NO: 34 or anucleic acid sequence that is 95% or more, 96% or more, 97% or more, 98%or more, or 99% or more homologous to the nucleic acid sequence of SEQID NO: 34; or an codon-optimized nucleic acid sequence of nucleic acids5052 to 6671 of SEQ ID NO: 34 or a nucleotide sequence that is 95% ormore, 96% or more, 97% or more, 98% or more, or 99% or more homologousto the nucleic acids 5052 to 6671 of SEQ ID NO:
 34. 13. The method ofclaim 12, wherein the intermediates and products are found in a Cannabisplant.
 14. The method of claim 12, wherein the products comprise atleast one of: cannabinoids, terpenoids, stilbenoids, flavonoids,phenolic amides, lignanamides, spermidine alkaloids, andphenylpropanoids.
 15. The method of claim 12, wherein the expressionsystem comprises at least one of: a vector, a cosmid, BAC, YAC, and aphage.
 16. The method of claim 12, wherein optimizing the modified hostcomprises: manipulating genes in the the modified host; silencing genesin the modified host; and amplifying genes in the modified host.
 17. Themethod of claim 12, wherein the modified host is a bacteria, plant,alga, or fungus.
 18. The method of claim 16, further comprises:controlling carbon flux in the modified host to yield a product ofinterest.
 19. The method of claim 17, wherein the modified host is S.cerevisiae, E. coli, P. pastorisis, N. crassa, S. pombe, R. palmatum, C.longa, O. sativa, A. arborescens, A. terrus, C. sativa, S. griseus, S.erythera, S. coelicolor, S. toxytruini, S. cellulosum, P. floresceus, A.orientalis, S. livedans, A. vinclandii, B. subtilis, M tuberculosis, M.xanthus, A. oryzae, A. niger, R. palmatum, H. serrata, R. rubra, L.erythrhizon, L. acetobutylicum, C. utilis, A. niger, C. glutamicum, or aspecies of bacillus.
 20. The method of claim 1, wherein the productderives from a sugar molecule.